EP0201882A2 - Polypeptide de prolactine de poisson et ses dérivés - Google Patents

Polypeptide de prolactine de poisson et ses dérivés Download PDF

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Publication number
EP0201882A2
EP0201882A2 EP86106359A EP86106359A EP0201882A2 EP 0201882 A2 EP0201882 A2 EP 0201882A2 EP 86106359 A EP86106359 A EP 86106359A EP 86106359 A EP86106359 A EP 86106359A EP 0201882 A2 EP0201882 A2 EP 0201882A2
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Prior art keywords
dna
prolactin
polypeptide
fish
keta
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EP86106359A
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German (de)
English (en)
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EP0201882A3 (fr
Inventor
Yoshihisa Kuwana
Tetsuro Kuga
Moriyuki Sato
Seiga Itoh
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KH Neochem Co Ltd
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Kyowa Hakko Kogyo Co Ltd
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Priority claimed from JP60099094A external-priority patent/JPS61257999A/ja
Priority claimed from JP60227531A external-priority patent/JPS6287600A/ja
Priority claimed from JP61075210A external-priority patent/JPS62234096A/ja
Application filed by Kyowa Hakko Kogyo Co Ltd filed Critical Kyowa Hakko Kogyo Co Ltd
Publication of EP0201882A2 publication Critical patent/EP0201882A2/fr
Publication of EP0201882A3 publication Critical patent/EP0201882A3/fr
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/57554—Prolactin
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli

Definitions

  • the present invention relates to a DNA coding for a fish prolactin polypeptide, for example, the prolactin polypeptide of Oncorhynchus keta, or a derivative thereof, a recombinant DNA wherein said DNA is incorporated, a microorganism containing the recombinant DNA and a process for the production of the fish prolactin polypeptide or a derivative thereof using the microorganism.
  • the fish prolactin and its derivatives have the effect of stimulating the growth of fish.
  • Oncorhynchus keta has adaptability to fresh water from the fact that when it is put in fresh water osmotic pressure of its plasma slightly decreases after 24 hours and is kept at a constant level thereafter.
  • Prolactin seems to have an important role in adaptation of Oncorhynchus keta to fresh water because the concentration of prolactin in blood increases significantly after 24 hours coincidentally with the decrease of osmotic pressure.
  • Mammalian prolactins are produced in the pituitary gland. The activity and structure of the mammalian prolactins are known. For example, human prolactin has been reported in Endocrinology 71, 209 (1962) by J. B. Josimovich, et al., and in Proc. Natl. Acad. Sci. USA 58, 2307 (1967) by L. M. Sherwood, et al.
  • rat prolactin gene [N. E. Cooke, et al., J. Biol. Chem. 255(13), 6502 (1980)]
  • bovine prolactin gene [J. H. Nilson, et al., Nucleic Acids Res., 8(7), 1561.(1980)]
  • human prolactin gene [N. E. Cooke, et al., J. Biol. Chem., 256(8), 4007 (1981)] are already known.
  • a fish prolactin gene has not been reported yet.
  • Fish prolactins have,the effect of stimulating,the growth of fish and the effect of adjusting the adaptability of fish to fresh water, and thus are useful as a component of baits for fish cultivation.
  • the amount of the prolactin provided by the recovery from the fish pituitary gland is limited. Therefore, it has been desired that a process for providing a large amount of fish prolactin at a low cost is developed.
  • the present inventors studied a method of producing fish prolactins by recombinant DNA techniques. As the result, the present inventors have successfully recovered a DNA complementary to a fish prolactin polypeptide and a derivative thereof which are usable in the production of the fish prolactin polypeptide and a derivative thereof, and produced a recombinant DNA and a microorganism containing the DNA. That is, a messenger RNA (mRNA) was extracted from the pituitary gland of Oncorh y nchus keta and a DNA (cDNA) complementary to the mRNA was synthesized.
  • mRNA messenger RNA
  • cDNA DNA complementary to the mRNA
  • the present inventors have further studied and found that a large amount of prolactin of Oncorh y nchus keta is formed and accumulated in a medium by culturing a microorganism containing a recombinant DNA wherein a DNA coding for the prolactin of Oncorhynchus keta' is incorporated. Further, the present inventors have studied and found that a large amount of a prolactin polypeptide derivative of Oncorhvnchus keta is formed and accumulated in a medium by culturing a microorganism containing a recombinant DNA wherein a DNA coding for the prolactin polypeptide derivative of Oncorhvnchus keta is incorporated.
  • the present invention provides a fish prolactin polypeptide, for example, the polypeptide having the peptide sequence as illustrated in Table 1 and a derivative thereof.
  • the polypeptide can be produced by recombinant DNA techniques in the following manner.
  • an mRNA of fish prolactin is used as a template to prepare a DNA (cDNA) complementary to the mRNA, and then a recombinant plasmid wherein the cDNA or a derivative thereof is incorporated is prepared. Further, the recombinant plasmid is incorporated in a host microorganism.
  • the DNA and recombinant plasmid can be used for amplification of a prolactin gene or a derivative thereof in bacteria such as Escherichia coli. Microorganisms carrying the recombinant plasmid are useful for producing a large amount of fish prolactin polypeptide and derivatives thereof at low cost.
  • the present invention provides a DNA coding for a fish prolactin polypeptide or a derivative thereof, a recombinant DNA wherein the DNA is incorporated, a microorganism containing the recombinant DNA and a process for producing the fish prolactin polypeptide and the derivative using the microorganism.
  • the DNA and recombinant plasmid of the present invention are prepared-by the following general method.
  • RNA is prepared from the pituitary gland of Oncorhynchus keta and passed through an oligo(dT) cellulose column to isolate RNA having polyadenylic acid [poly(A)] [poly(A)RNA].
  • a double stranded DNA is synthesized using the poly(A)RNA as a template and a reverse transcriptase.
  • a recombinant is obtained using in vitro recombinant DNA techniques by inserting the synthetic DNA into a vector DNA such as Escherichia coli plasmid DNA.
  • a recombinant plasmid having the DNA complementary to the prolactin mRNA of Oncorhvnchus keta is selected based on the marker carried on the plasmid.
  • a DNA coding for a fish prolactin polypeptide derivative is obtained by treating the DNA coding for the fish prolactin polypeptide with an enzyme.
  • the DNA is inserted into a vector DNA to obtain a recombinant DNA and the recombinant DNA is incorporated into a microorganism to obtain a transformant.
  • a fish prolactin polypeptide derivative is obtained by culturing the transformant.
  • the pituitary gland is excised from captured Oncorhynchus keta and immediately freezed in a liquid nitrogen.
  • Guanidium isothiocyanate is added to the freezed pituitary gland and the pituitary gland is disrupted and solubilized. Then, the solubilized pituitary gland is put on CsCl solution layer and subjected to ultra centrifugation to obtain whole cytoplasmic RNA as a precipitate.
  • LiCl is added to the solubilized matter with guanidium isothiocyanate to recover only RNA as a precipitate.
  • the extracted RNA is dissolved in an NaCl or KC1 hypertonic solution (for example 0.5M) and passed through an oligo(dT) cellulose clumn to allow mRNA having poly(A) to be adsorbed on the column. Elution is carried out with water or a hypotonic salt solution such as 10 mM Tris-HCl buffer to isolate the mRNA having poly(A).
  • an NaCl or KC1 hypertonic solution for example 0.5M
  • oligo(dT) cellulose clumn for example 0.5M
  • Elution is carried out with water or a hypotonic salt solution such as 10 mM Tris-HCl buffer to isolate the mRNA having poly(A).
  • Synthesis of cDNA and insertion of the cDNA into a vector are carried out according to the method of Okayama-Berg [Okayama & Berg; Mol. Cell. Biol. 2, 161 (1982)] as described below.
  • a vector primer is synthesized.
  • a vector e.g. pCDVl
  • Kpnl is treated with Kpnl in an adequate solution such as a solution consisting of Tris-HCl buffer (e.g. pH 7.5, 10 mM), MgCl 2 (e.g. 6 mM) and NaCl (e.g. 10 mM) to cut pCDVl at KpnI site.
  • the DNA is incubated with terminal deoxynucleotidyltransferase at an appropriate temperature (e.g. 37°C) for an appropriate period (e.g. 20 minutes) in a solution consisting of Tris-HC1 buffer (e.g. pH 6.8, 30 mM), sodium cacodylate (e.g.
  • the DNA is cut with EcoRI in a solution consisting of Tris-HC1 buffer (e.g. pH 7.5, 10 mM), MgCl 2 (e.g. 6 mM) and NaCl (e.g. 100 mM).
  • Tris-HC1 buffer e.g. pH 7.5, 10 mM
  • MgCl 2 e.g. 6 mM
  • NaCl e.g. 100 mM
  • the digested solution is fractionated by low-gelling-temperature agarose gel electrophoresis [Lars Wieslander: Analytical Biochemistry, 98, 305 (1979), hereinafter referred to 'as LGT method] to recover a DNA fragment of about 3.1 Kb.
  • the DNA is dissolved in an NaCl or KC1 hypertonic solution (e.g. 0.5M) and pas.sed through a poly(dA) cellulose column to allow only vector primer molecules having poly(T) to be adsorbed on the column. Elution is carried out with water or a hypotonic salt solution such as 10 mM Tris-HCl buffer to isolate only the vector primer molecules with poly(T).
  • a linker DNA is synthesized in the following manner.
  • pLl DNA is treated with PstI in an appropriate solution such as a solution consisting of Tris-HC1 buffer (e.g. pH 7.5, 10 mM), MgCl 2 (e.g. 6 mM) and NaCl (e.g. 50 mM) to cut pLl at PstI site.
  • the DNA is treated by the same method as in the synthesis of the vector primer except that dGTP is added in place of dTTP, and oligo dG chains (about 15 mer) are added.
  • the DNA is cut with HindIII in an appropriate solution such as a solution consisting of Tris-HCl buffer (e.g.
  • a DNA fragment of about 0.5 Kb is fractionated by agarose gel electrophoresis and recovered with DEAE paper. Thus, a linker DNA is obtained.
  • poly(A)RNA, vector primer and linker DNA are used to synthesize cDNA in the following manner.
  • the poly(A)RNA and vector primer DNA are reacted with a reverse transcriptase at an appropriate temperature (e.g. 37°C) for an appropriate period (e.g. 40 minutes) in a solution consisting of Tris-HCl buffer (e.g. pH 8.3, 50 mM), MgCl 2 (e.g. 8 mM), KC1 (e.g. 30 mM), dithiothreitol (e.g. 0.3 mM), and dATP, dTTP, dCTP and dGTP (e.g. 2 mM each).
  • Tris-HCl buffer e.g. pH 8.3, 50 mM
  • MgCl 2 e.g. 8 mM
  • KC1 e.g. 30 mM
  • dithiothreitol e.g. 0.3 mM
  • dATP
  • Oligo dC chains (about 15 mer) are added to the 3' ends of the thus obtained RNA-DNA double stranded chain under the same conditions as in the case of the addition of dT chains to the vector primer except that dTTP is replaced with dCTP.
  • the DNA is cut with HindIII in a solution consisting of Tris-HCl buffer (e.g. pH 7.5, 10 mM), MgCl 2 (e.g. 6 mM) and NaCl (e.g. 60 mM).
  • the previously prepared linker DNA is mixed with the DNA and the mixture is incubated with Escherichia coli DNA ligase at an appropriate temperature (e.g. 12°C) for an appropriate period (e.g.
  • Tris-HC1 buffer e.g. pH 7.5, 20 mM
  • MgC1 2 e.g. 4 mM
  • (NH 4 ) 2 SO 4 e.g. 10 mM
  • KC1 e.g. 0.1M
  • ⁇ -nicotinamide adenine dinucleotide (0-NAD) e.g. 0.1 mM
  • Escherichia coli DNA ligase, Escherichia coli DNA polymerase I and Escherichia coli ribonuclease H are added to replace the RNA part with DNA and to obtain a recombinant plasmid containing a complete double stranded cDNA.
  • Escherichia coli strain e.g. Escherichia coli c600SF8 is transformed with the thus obtained recombinant plasmid, for example, by the method of Scott, et al. [Katsuya Shigesada: Saibo Kogaku (Cell Engineering), 2, 616 (1983)]. Since an ampicillin resistance gene exists in the recombinant plasmid mentioned above, the Escherichia coli transformant is resistant to ampicillin.
  • Selection of a microorganism strain carrying a new recombinant plasmid DNA having a gene complementary to the mRNA of fish prolactin from the ampicillin-resistant (Ap R ) strains is generally carried out in the following manner. That is, the transformants obtained above are fixed on.a nitrocellulose filter and a synthetic DNA probe having a DNA sequence which is presumed from the amino acid sequence of a known prolactin of Oncorh y nchus keta is'hybridized thereto to select the transformant showing strong hybridizaion [the method of Grunstein-Hogness, Proc. Natl. Acad. Sci., USA., 72, 3961 (1975)].
  • the probe DNA is synthesized by a conventional triester method [J. Am. Chem. Soc., 97, 7327 (1975)]. Selection by the synthesized DNA probe is more definitely carried out by the method of Southern, et al. [J. Mol. Biol., 98, 503 (1975)] and a recombinant plasmid DNA having the gene complementary to Oncorhynchus keta prolactin mRNA is identified by the same method.
  • pSPRLl is an example of the thus obtained recombinant plasmids.
  • the plasmid can be used as a source of the DNA coding for prolactin of Oncorhynchus keta.
  • the DNA coding for prolactin of Oncorhynchus keta is cut out from the plasmid carrying the DNA and inserted into a vector DNA.
  • the thus obtained recombinant DNA is incorporated in a microorganism and the obtained transformant is cultured to accumulate a prolactin polypeptide of Oncorhvnchus keta in a medium, which is then recovered. In this manner, the prolactin polypeptide of Oncorhvnchus keta can be produced.
  • Plasmid pSPRLl can be recovered from Escherichia coli ESPRL1 (FERM BP-776) containing the plasmid by the method of Birnboim [Nucleic Acids Res. 7, 1513 (1979)].
  • a DNA coding for a prolactin polypeptide derivative is obtained by treating the DNA coding for prolaction of Oncorhynchus keta with an enzyme. Plasmid pSPRLl is used as a source of the DNA coding for prolactin of Oncorhvnchus keta.
  • any vector can be used so long as the DNA incorporated therein can be expressed in a microorganism.
  • a vector DNA wherein a DNA can be inserted downstream from a suitable promoter such as tryptophan (trp) promoter, lactose (lac) promoter or PL promoter and the length between Shine-Dalgarno sequence (referred to as SD sequence hereinafter) and initiation codon (ATG) is adjusted, for example, to 6 - 18 base pairs is employed.
  • trp tryptophan
  • lac lactose
  • ATG initiation codon
  • Preferred examples of the vector DNA are pGELl, pTrS20 (Japanese Patent Appliction No. 23319/86) and pKYP26.
  • pGELl is a plasmid as illustrated in Fig.
  • Plasmid pTrS20 is obtained according to the method described in Reference Example 1.
  • a microorganism containing plasmid pKYP26 was deposited with the FRI as Escherichia coli IKYP26 (FERM BP-863) on Auqust 8, 1985.
  • Plasmid pKYP26 can be obtained from Escherichia coli IKYP26 (FERM BP-863) containing the plasmid by the method of Birnboim.
  • Recombination of the DNA coding for the polypeptide and the vector DNA can be carried out by a conventional recombinant DNA techniques wherein both DNAs are digested with restriction enzymes and religated with T4 DNA ligase.
  • the DNA fragments and synthetic DNA linker äescribed above are ligated with T4 DNA ligase to obtain the recombinant plasmid pSPRLA6 illustrated in Fig. 3.
  • BanIII-BamHI digestion fragment coding for the mature prolactin peptide of Oncorhvnchus keta is obtained from pSPRLA6, and BanIII-BamHI digestion fragment containing a tryptophan promoter is obtained from pGELI.
  • the two DNA fragments are ligated with T4 DNA ligase to obtain the recombinant plasmid pSPRLBI illustrated in Fig. 3.
  • the plasmid has a construction wherein a region coding for mature prolactin of Oncorhynchus keta is ligated downstream from the tryptophan promoter.
  • a recombinant plasmid is obtained by the following method.
  • Plasmid pSPRLl is subjected to cleavage with KpnI, digestion with Ba131 and cleavage with BamHI to obtain a DNA fragment containing a DNA codig for prolactin polypeptide.
  • pKYP26 is cleaved with BamHI and PstI to obtain a DNA fragment of about 1,700 bp containing lpp terminator region.
  • Plasmid pTrS20 is subjected to cleavage with SacI, formation of a flush end with DNA polymerase I and cleavage with PstI to obtain a DNA fragment of about 1,000 bp containing trp promoter.
  • a recombinant plasmid is prepared using T4 DNA ligase.
  • Escherichia coli HB101 is transformed with the recombinant plasmid.
  • a colony resistant to ampicillin (Ap R ) is obtained and a plasmid DNA is recovered from the colony.
  • Cleavage maps of the thus obtained plasmids pSPRLQ28 and pSPRLQ52 are illustrated in Fig. 4.
  • Reaction conditions required for the recombinant DNA techniques described above are generally as follows.
  • Digestion of the DNA with restriction enzymes is usually carried out by reacting 0.1 to 20 ⁇ g of the DNA with 0.1 - 100 units, preferably 1 - 3 units of the restriction enzyme per 1 ug of the DNA in a mixture of 2 - 200 mM, preferably 10 - 40 mM Tris-HCl (pH 6.0 - 9.5, preferably pH 7.0 - 8.0), 0 - 200 mM NaCl and 2 - 30 mM, preferably 5 - 10 mM MgC1 2 at 20 -70°C (optimal temperature depends on the restriction enzymes used) for 15 minutes to 24 hours. Reaction is usually stopped by heating at 55 - 75°C for 5 - 30 minutes, or alternatively by inactivating the restriction enzyme with a reagent such as phenol or diethylpyrocarbonate.
  • a reagent such as phenol or diethylpyrocarbonate.
  • Purification of the DNA fragments formed by digestion with restriction enzymes is carried out by LGT method or polyacrylamide gel electrophoresis.
  • Ligation of the DNA fragments is carried out with 0.3 - 10 units of T4 DNA ligase in a mixture of 2 - 200 mM, preferably 10 - 40 mM Tris-HCl (pH 6.1 - 9.5, preferably 7.0 - 8.0), 2 - 20 mM, preferably 5 - 10 mM MgCl 2 , 0.1 - 10 mM preferably 0.5 - 2.0 mM ATP and 1 - 50 mM, preferably 5 - 10 mM dithiothreitol at 1 - 37°C, preferably 3 - 20°C for 15 minutes to 72 hours, preferably 2 - 20 hours.
  • the recombinant plasmid DNA formed by the ligation reaction is introduced into Escherichia coli by the transformation method of Cohen, et al. [S.N. Cohen, et al.: Proc. Natl. Acad. Sci. USA, 69, 2110 (1972)], if necessary.
  • Isolation of the recombinant plasmid DNA from Escherichia coli carrying the DNA is carried out by the method described in Example 1 or the method of Birnboim, et al. [H.C. Birnboim, et al.: Nucleic Acids Res. 7, 1513 (197.9)].
  • Plasmid DNA is digested with 1 - 10 kinds of restriction endonucleases and the cleavage sites are examined by agarose gel electrophoresis or polyacrylamide gel electrophoresis. Further, if necessary, the base sequence of the DNA is determined by the method of Maxam-Gilbert [Proc. Natl. Acad. Sci. 74, 560 (1977)] or the method of Sanger [Sanger, et al.: Proc. Natl. Acad. Sci. USA, 74, 5463 (1977); Amersham Co., M13 cloning and sequencing handbook] using M13 phage.
  • the fish prolactin polypeptide and derivatives thereof of the present invention are produced by the following method.
  • Escherichia coli K-12 HB101 is transformed with a plasmid such as pSPRLBl, pSPRLQ28 or pSPRLQ52 and an Escherichia coli strain carrying the plasmid is selected from the ampicillin resistant colonies.
  • the Escherichia coli strain carrying the plasmid is cultured in a medium to produce the fish prolactin polypeptide or a derivative thereof in the cultured product.
  • the medium either a synthetic medium or a natural medium can be used so long as it is suitable for the growth of Escherichia coli and the production of the fish prolactin polypeptide.
  • glucose fructose, lactose, glycerol, mannitol, sorbitol, etc. may be used.
  • NH 4 C1 As the nitrogen source, NH 4 C1, (NH 4 ) 2 S0 4 , casamino acid, yeast extract, polypeptone, meat extract, Bacto-Tryptone, corn steep liquor, etc. may be used.
  • nutrients such as K 2 HP0 4 , KH 2 PO 4 , NaCl, MgS0 4 , vitamine B l and MgCl 2 may be used.
  • Culturing is carried out at pH 5.5 - 8.5 and at 18 - 40°C with aeration and stirring.
  • the prolactin polypeptide of Oncorhynchus keta or the derivative thereof is accumulated in cultured cells.
  • the collected cells are treated with lysozyme, disrupted by repeated freezing and thawing and subjected to centrifugation.
  • the thus obtained supernatant fluid is subjected to extraction according to a conventional method for extraction of polypeptides to recover the polypeptide.
  • Detection of the polypeptide is carried out by heat-dissolving the cultured cells directly in Sample buffer of Laemmli [Laemmli, Nature, 227, 680 (1970)] and by subjecting the solution to SDS-polyacrylamide gel (the method of Laemmli: the reference mentioned above) and Coomassie Brilliant Blue staining.
  • RNA having poly(A) was prepared from the pituitary gland of Oncorh y nchus keta according to guanidium thiocyanate-cesium chloride method [edited by Maniatis, et al.: Molecular Cloning, p196, published by Cold Spring Harbor; Katsuya Shigesada, Saibo Kogaku (Cell Engineering), 2, 616 (1983)] in the following manner.
  • RNA precipitate was dissolved in 10 ml of Tris-HCl solution (pH 8.0).containing 1 mM EDTA. After extraction with phenol-chloroform, the RNA was recovered with ethanol as a precipitate. About 1 mg of the thus obtained RNA was dissolved in 1 ml of a solution consistig of 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA. The solution was incubated at 65°C for 5 minutes and 0.1 ml of 5M NaCl was added.
  • the mixture was subjected to oligo(dT) cellulose column (product of P-L Biochemicals, column volume 0.5 ml) chromatography.
  • the mRNA having poly(A) adsorbed on the column was eluted with a solution consisting of 10 mM Tris-HCl (pH 7.5) and 1 mM EDTA and fractionated in 0.2 ml portions to obtain about 10 ug of the mRNA having poly(A) in the 3rd to 5th fractions.
  • pCDVl [Okayama & Berg: Mol. Cell. Biol., 3, 280 (1983)] was added to 300 ⁇ l of a solution consisting of 10 mM Tris-HCl (pH 7.5), 6 mM MgCl 2 and 10 mM NaCl, and further 500 units of KpnI (product of Takara Shuzo Co., the restriction enzymes used hereinafter are all products of Takara Shuzo Co., unless otherwise specified) was added. Reaction was carried out at 37°C for 6 hours to cut the plasmid at KpnI site. After phenol-chloroform extraction, a DNA was recovered by ethanol precipitation.
  • TdT buffer a buffer consisting of 40 mM sodium cacodylate, 30 mM Tris-HCl (pH 6.8), 1 mM CaCl 2 and 0.1 mM dithiothreitol (referred to as DTT hereinafter).
  • TdT terminal deoxynucleotidyl transferase
  • the reaction product was subjected to LGT method to obtain a DNA fragment of about 3.1 Kb which is about 60 ⁇ g of pCDVl with poly(dT) chains.
  • the DNA was dissolved in 500 ml of a solution consisting of 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA. The solution was incubated at 65°C for 5 minutes and 50 ⁇ l of 5M NaCl was added under ice cooling. The mixture was subjected to oligo(dA) cellulose column (product of Collaborative Research) chromatography. The DNA with enough poly(dT) chains was adsorbed on the column.
  • linker DNA was prepared in the following manner.
  • pL1 [Okayama & Berg: Mol. Cell. Biol., 3, 280 (1983)] was added to 200 ⁇ l of a buffer consisting of 10 mM Tris-HCl (pH 7.5), 6 mM MgCl 2 and 50 mM NaCl. Further, 50 units of pstI was added and reaction was carried out at 37°C for 4 hours to cut pLl DNA at PstI site. The reaction product was subjected to phenol-chloroform extraction and ethanol precipitation to recover about 13 ⁇ g of pLl DNA cleaved with PstI.
  • the reaction product was fractionated by agarose gel electrophoresis and a DNA fragment of about 0.5 Kb was recovered by DEAE paper method [Dretzen, et al., Anal. Biochem., 112, 295 (1981)] .
  • the DNA was linker DNA with cligo (dG) chain (referred to as linker-DNA hereinafter) .
  • RNA and about 1.4 ⁇ g of the vector primer prepared W were dissolved in 22.3 ⁇ l of a solution consisting of 50 mM Tris-HCl (pH 8.3), 8 mM MgCl 2 , 30 mM KC1, 0.3 mM DTT, 2 mM dNTP (dATP, dTTP, dGTP and dCT P ) and 10 units of ribonuclease inhibitor (product of P-L Biochemicals).
  • Ten units of reverse transcriptase (product of Seikagaku Kogyo Co.) was added and incubation was carried out at 37°C for 40 minutes to synthesize a DNA complementary to the mRNA.
  • the reaction product was subjected to phenol-chloroform extraction and ethanol precipitation to recover a vector-primer DNA associated with RNA-DNA double stranded chain.
  • the DNA was dissolved in 20 ⁇ l of TdT buffer containing 66 ⁇ M dCTP and 0.2 ⁇ g of poly(A).
  • TdT product of P-L Biochemicals
  • Fourteen units of TdT was added and incubation was carried out at 37°C for 8 minutes to add (dC) chains (12 mer) to the 3' ends of the cDNA.
  • the reaction product was subjected to phenol-chloroform extraction and ethanol precipitation to recover a cDNA-vector primer DNA associated with (dC) chains.
  • the DNA was dissolved in 400 ul of a solution consisting of 10 mM Tris-HCl (pH 7.5), 6 mM MgCl 2 and 60 mM NaCl. Twenty units of HindIII was added and incubation was carried out at 37°C for 2 hours to cut the DNA at HindIII site. The reaction product was subjected to phenol-chloroform extraction and ethanol precipitation to obtain 0.5 pmole of a cDNA-vector primer DNA associated with (dC) chains.
  • 0.08 pmole of the DNA and 0.16 pmole of the linker-DNA mentioned above were dissoved in 40 ⁇ l of a solution consisting of 10 mM Tris-HC1 ⁇ (pH 7.5), 0.1M NaCl and 1 mM EDTA and incubations were carried out at 65°C, 42°C and 0°C for 10 minutes, 25 minutes and 30 minutes, respectively.
  • the reaction solution was adjusted to 400 ⁇ l (total volume) of a solution having a composition of 20 mM Tris-HCl (pH 7.5), 4 mH McCl 2 , 10 mM (Nn 4 ) 2 SO 4 , 0.1M KCl and 0.1 mM ⁇ -NAD.
  • Escherichia coli DNA ligase (product of New England Biolabs, was addes to the reaction solution and incubation was carriea out at 11°C overnight.
  • the reaction solution was adjusted to a solution containing 40 ⁇ M dNTP and 0.15 mM 8-NAD.
  • Five units of Escherichia coli DNA ligase, 7 units of Escherichia coli DNA polymerase I (product of P-L Biochemicals) and 2 units of Escherichia coli ribonuclease H (product of P-L Biochemicals) were added and incubation was carried out at 12°C for one hour and successively at 25° C for one hour.
  • a recombinant DNA containing the cDNA was cyclized, the RNA part of the RNA-DNA double stranded chain was replaced with the DNA and a recombinant plasmid having complete double stranded DNA was formed.
  • plasmid pSPRLl containing a DNA coding for prolactin of Oncorhvnchus keta was dissolved in 100 ⁇ l of a solution containing 10 mM Tris-HCl (pH 7.5), 7 mM MgCl 2 , and 100 mM NaCl (referred to as Y-100 buffer solution hereinafter). Then, 30 units of restriction enzyme HindIII (product of Toyo Boseki Co.) was added and digestion reaction was carried out at 37°C for 2 hours.
  • HindIII product of Toyo Boseki Co.
  • a DNA fragment of about 0.7 Kb containing N-terminal region, 5'-non-translational region of'the prolactin and the vector part encoded by pSPRLl was obtained from the reaction solution by LGT method.
  • the DNA of 0.7 Kb was dissolved in 20 ⁇ l Y-100 buffer solution and 8 units of HaeIII was added. Digestion reaction was carried out at 37°C for 2 hours.
  • About 0.1 ⁇ g of a DNA fragment of 43 bp corresponding to N-terminal region was obtained from the reaction solution by polyacrylamide gel electrophoresis and filtration with glass filter.
  • pSPRLl was dissolved in 30 ⁇ l of Y-100 buffer solution. Eight units of BanIII and 16 units of HindIII were added and digestion reaction was carried out at 37°C for 2 hours. About 1.0 ⁇ g of a DNA fragment of about 3.7 Kb containing C-terminal region, 3'-non-translational region of Oncorhynchus keta prolactin and vector part encoded by pSPRLl was obtained from the reaction solution by LGT method.
  • Escherichia coli K294 was transformed using the reaction solution to obtain an Ap R colony.
  • a plasmid DNA pSPRLA6 illustrated in Fig. 3 was recovered from the colony.
  • the structure of pSPRLA6 was recognized by the cleavage with PstI, BamHI, EcoRI, BanIII, BglII and HindIII and agarose gel electrophoresis.
  • pGELl 2 ⁇ g was dissolved in 40 ⁇ l of Y-100 buffer solution and 10 units each of BamHI and BanIII were added. Digestion reaction was carried out at 37°C for 3 hours. About 0.2 ⁇ g of a DNA fragment of about 2.7 Kb containing a tryptophan promoter was obtained from the reaction solution by LGT method.
  • Escherichia coli K294 was transformed using the reaction solution to obtain an Ap R colony.
  • a plasmid DNA pSPRLBl illustrated in Fig. 3 was recovered from the colony.
  • the structure of pSPRLB1 was recognized by the cleavage with EcoRI, HindIII, BanIII, PstI and BamHI and agarose gel electrophoresis.
  • Escherichia coli containing plasmid pSPRLBl was deposited with the FRI as Escherichia coli ESPRLB1 under FERM BP-777 on April 26, 1985.
  • chloroform extraction method This step is referred to as chloroform extraction method.
  • ethanol precipitation method One tenth volume of 3M sodium acetate was added and 2.5 times volume of ethanol was added. The mixture was allowed to stand at -20°C for 2 hours and a precipitate was recovered by cooling centrifugation (4°C, 11,000 rpm, 10 minutes). This step is referred to as ethanol precipitation method.
  • the precipitate was dissolved in 50 ⁇ l of a solution consisting of 20 mM Tris-HCl (pH 8.0), 12 mM CaC1 2 , 12 mM MgC1 2 , 200 mM NaCl and 1 mM EDTA. The solution was kept at 20°C for 5 minutes. Then, 10 units of BAL31 nuclease (product of Bethesda Research Laboratories Inc.) was added and decomposition reaction was carried out at 20°C for 20 minutes. After the reaction, phenol extraction and chloroform extraction were carried out and a DNA was recovered by ethanol precipitation.
  • BAL31 nuclease product of Bethesda Research Laboratories Inc.
  • the DNA was dissolved in 40 ⁇ l of a solution consisting of 10 mM Tris-HCl (pH 7.5), 7mM MgCl 2 , ' 100 mM NaCl and 6 mM mercaptoethanol (referred to as Y-100-2 buffer solution hereinafter) and 10 units of BamHI was added. Digestion reaction was carried out at 37°C for 2 hours. From the reaction solution, about 0.5 ug of a DNA fragment of about 1.2 Kb containing a DNA coding'for Oncorhynchus keta prolactin polypeptide was obtained by LGT method.
  • pKYP26 3 ⁇ g was dissolved in 40 ⁇ l of Y-100-2 buffer solution, and-8 units of BamHI and 10 units of PstI were added. Digestion reaction was carried out at 37°C for 2 hours. From the reaction solution, 0.3 ⁇ g of a DNA fragment of about 1,700 base pairs (bp) containing lpp terminator part was obtained by LGT method.
  • pTrS20 3 ug of pTrS20 was dissolved in 40 ⁇ l of Y-0 buffer solution. 10 units of SacI was added and digestion reaction was carried out at 37°C for 2 hours. After phenol extraction and chloroform extraction of the reaction solution, a DNA was recovered by ethanol precipitation. The DNA was dissolved in 30 ⁇ l of a solution consisting of 50 mM Tris-HCl (pH 7.5), 5 mM MgCl 2 , 10 mM mercaptoethanol and 0.4 mM each of dATP, dCTP, dGTP and dTTP. 4 units of DNA polymerase I (product of Takara Shuzo Co.) was added and reaction was carried out at 20°C for one hour.
  • DNA polymerase I product of Takara Shuzo Co.
  • a DNA was recovered by ethanol precipitation.
  • the DNA was dissolved in 30 ⁇ l of Y-100-2 buffer solution and 6 units of PstI was added. Digestion reaction was carried out at 37°C for 2 hours. From the reaction solution, 0.1 ⁇ g of a DNA fragment of about 1,000 bp containing a trp promoter was obtained by LGT method.
  • T4 DNA ligase buffer solution 3 units of T4 DNA ligase (product of Takara Shuzo Co.) was added and ligation reaction was carried out at 4°C for 16 hours.
  • Escherichia coli HB101 [Bolivar et. al., Gene, 2, 75 (1977)] was transformed using the reaction solution and colonies showing Ap R were obtained. Plasmid DNAs pSPRL Q 28 and pSPRLQ52 illustrated in Fig. 4 were recovered from the colonies by the method of Birnboim, et. al. [Nucleic Acids Research, 7, 1513 (1979)].
  • pSPRLQ28 had a structure coding for a prolactin polypeptide derivative wherein the DNA coding for four amino acids at the N-terminal of Oncorhvnchus keta prolactin polypeptide was lost and methionine derived from pTrS20 was attached to the N-terminal amino acid (aspartic acid) of the remaining polypeptide.
  • pSPRLQ52 had a structure coding for a prolactin polypeptide derivative wherein the DNA coding for nine amino acids at the N-terminal was lost and methionine derived from pTrS20 was attached to the N-terminal amino acid (alanine) of the remaining polypeptide.
  • Nucleotide sequences of the genes of Oncorhvnchus keta prolactin polypeptide derivatives in pSPRLQ28 and pSPRLQ52 and amino acid sequences of the polypeptide derivatives are illustrated in Table 2 and Table 3, respectively.
  • Escherichia coli containing plasmids pSPRLQ28 and pSPRLQ52 were deposited with the FRI as Escherichia coli ESPRLQ28 (FERM BP-1006) and Escherichia coli ESPRLQ52 (FERM BP-1007) on March 28, 1986.
  • the microorganisms which produce prolactin polypeptide derivatives of Oncorh y nchus keta were cultured according to Example 8.
  • the cells were collected by centrifugation at 8,000 rpm for 10 minutes and washed with a buffer solution (pH 7.5) containing 30 mM NaCl and 30 mM Tris-HC1.
  • the washed cells were suspended in 5 m£ of the buffer mentioned above and subjected to ultrasonic disintegration (Branson Sonic Power Company, Sonifier cell disruptor 200, output cotrol treatment for 2 and 10 minutes).
  • Disrupted cells were recovered by centrifugation at 15,000 rpm for 30 minutes.
  • the disrupted cells were treated according to the method of Marston, et al. [F.A.O.
  • Synthetic DNAs of 19-mer and 17-mer (10 pmoles each) were dissolved in 20 ⁇ l (total volume) of a solution containing 50 mM Tris-HC1 (pH 7.5), 10 mM MgCl 2 , 5 mM dithiothreitol, 0.1 mM EDTA and 1 mM ATP and 3 units of T4 polynucleotide kinase (product of Takara Shuzo Co.) was added. Phosphorylation reaction was carried out at 37°C for 60 minutes.
  • Escherichia coli HB101 [Bolivar, et al..: Gene 2, 75 (1979)] was transformed with the thus obtained mixture of the recombinant plasmids. Ap R colonies were obtained and a plasmid DNA was recovered from the cultured cells of the colonies. The structure of the thus obtained plasmid was confirmed by cleavage with EcoRI, BanIII, HindIII,' SacI and NruI and agarose gel electrophoresis. The plasmid was named pTrS20 (Fig. 5). It was confirmed by the method of Sanger using M13 phage that the nucleotide sequence around the BanIII and HindIII sites of pTrS20 was as follows.
EP86106359A 1985-05-10 1986-05-09 Polypeptide de prolactine de poisson et ses dérivés Withdrawn EP0201882A3 (fr)

Applications Claiming Priority (6)

Application Number Priority Date Filing Date Title
JP99094/85 1985-05-10
JP60099094A JPS61257999A (ja) 1985-05-10 1985-05-10 魚類のプロラクチンポリペプチド
JP60227531A JPS6287600A (ja) 1985-10-12 1985-10-12 魚類のプロラクチンポリペプチド
JP227531/85 1985-10-12
JP61075210A JPS62234096A (ja) 1986-04-01 1986-04-01 魚類のプロラクチンポリペプチド誘導体
JP75210/86 1986-04-01

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0387457A1 (fr) * 1989-01-06 1990-09-19 Eurogentec S.A. Hormones protéiniques recombinantes de poisson
US5320952A (en) * 1989-09-21 1994-06-14 W. R. Grace & Co.-Conn. Enhanced gene expression in response to lactation signals

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
CHEMICAL ABSTRACTS, vol. 100, no. 5, 30th January 1984, page 167, abstract no. 31108d, Columbus, Ohio, US; P. PRUNET et al.: "Purification and biological characterization of chinook salmon prolactin", & GEN. COMP. ENDOCRINOL. 1984, 53(1), 49-57 *
CHEMICAL ABSTRACTS, vol. 98, no. 11, 14th March 1983, page 204, abstract no. 85081y, Columbus, Ohio, US; H. KAWAUCHI et al.: "Isolation and properties of chum salmon prolactin", & GEN. COMP. ENDOCRINOL. 1983, 49(3), 446-58 *
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE USA, vol. 82, no. 22, November 1985, pages 7490-7494, Washington, US; J.L. SPECKER et al.: "Isolation and partial charcterization of a pair of prolactins released in vitro by the pituitary of a cichlid fish, Oreochromis mossambicus" *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0387457A1 (fr) * 1989-01-06 1990-09-19 Eurogentec S.A. Hormones protéiniques recombinantes de poisson
US5320952A (en) * 1989-09-21 1994-06-14 W. R. Grace & Co.-Conn. Enhanced gene expression in response to lactation signals

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EP0201882A3 (fr) 1987-08-12

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